The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] builds and repairs the 3' terminal CCA sequence of all tRNAs by adding one nucleotide at a time, using CTP and ATP as substrates. Unlike all other sequence-specific RNA and DNA polymerases, the CCA-adding enzyme does not use a nucleic acid template. Thus the protein itself must serve as a template, or the enzyme must use a novel mechanism to specify nucleotide addition. We have shown that the CCA-adding enzyme has only a single active site, that the enzyme binds primarily to the acceptor stem ("top half") of tRNA, and that the tRNA remains immobile on the enzyme surface during addition of CCA. To explain how three nucleotides can be added to tRNA without movement of either the tRNA or the active site, we proposed that the growing 3' terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single nucleotide binding site. The ATP binding site would be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would therefore be a dynamic ribonucleoprotein structure, in a mechanism we call collaborative templating. Here we propose to study the CCA-adding enzyme in biochemical detail. The experiments will test the collaborative templating model, and provide a wealth of new information about the CCA-adding enzyme. Specifically, we will use photochemical crosslinking and hydroxyl radical footprinting to identify amino acid residues in the immediate vicinity of the active site, the nucleotide binding pocket, and the tRNA binding site; we will ask whether mutations in these residues change the specificity of CCA addition as predicted by the model; we will use nucleotide analogues to define the nature of the nucleotide binding pocket; and we will continue our efforts to crystallize or cocrystallize the CCA- adding enzyme with tRNA substrates. In principle, cocrystal structures of the enzyme with the three substrates (tRNA-N, tRNA- NC, tRNA-NCC) and the mature tRNA product (tRNA-NCCA where N is the "discriminator base") would provide a moving picture of this unusual enzyme in action.